Hedgehog Signaling Controls Dorsoventral Patterning, Blastema Cell Proliferation and Cartilage Induction During Axolotl Tail

Research article 3243 Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration Esther Schnapp1, Martin Kragl1, Lee Rubin2 and Elly M. Tanaka1,* 1Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany 2Curis Incorporated, 61 Moulton Street, Cambridge, MA 02138, USA *Author for correspondence (e-mail: [email protected])

Accepted 12 May 2005

Development 132, 3243-3253 Published by The Company of Biologists 2005 doi:10.1242/dev.01906

Summary Tail regeneration in urodeles requires the coordinated Proliferation of tail blastema cells was severely impaired, growth and patterning of the regenerating tissues types, resulting in an overall cessation of tail regeneration, and including the spinal cord, cartilage and muscle. The blastema cells no longer expressed the early cartilage dorsoventral (DV) orientation of the spinal cord at the marker Sox9. Spinal cord removal experiments revealed amputation plane determines the DV patterning of the that hedgehog signaling, while required for blastema regenerating spinal cord as well as the patterning of growth is not sufficient for tail regeneration in the absence surrounding tissues such as cartilage. We investigated this of the spinal cord. By contrast to the cyclopamine effect on phenomenon on a molecular level. Both the mature and tail regeneration, cyclopamine-treated regenerating limbs regenerating axolotl spinal cord express molecular markers achieve a normal length and contain cartilage. This study of DV progenitor cell domains found during embryonic represents the first molecular localization of DV patterning neural tube development, including Pax6, Pax7 and Msx1. information in mature tissue that controls regeneration. Furthermore, the expression of Sonic hedgehog (Shh) is Interestingly, although tail regeneration does not occur localized to the ventral floor plate domain in both mature through the formation of somites, the Shh-dependent and regenerating spinal cord. Patched1 receptor expression pathways that control embryonic somite patterning and indicated that hedgehog signaling occurs not only within proliferation may be utilized within the blastema, albeit

Development the spinal cord but is also transmitted to the surrounding with a different topography to mediate growth and blastema. Cyclopamine treatment revealed that hedgehog patterning of tail tissues during regeneration. signaling is not only required for DV patterning of the regenerating spinal cord but also had profound effects on Key words: Axolotl, Regeneration, Sonic hedgehog, Cyclopamine, the regeneration of surrounding, mesodermal tissues. Blastema, Sox9, Pax7

Introduction by Holtzer strongly suggests that cartilage is induced by the Axolotl tail regeneration involves the regrowth and patterning ventral half of the spinal cord during urodele tail regeneration. of multiple tissue types, including the spinal cord, muscle, It also implies that the mature spinal cord maintains DV cartilage, dermis, fin and skin. After tail amputation and patterning information and that this patterning information is epithelial wound healing, the spinal cord grows out as a tube transmitted into the regenerate. Similarly, studies in limb of neuroepithelial progenitor cells, called the ependymal tube. regeneration indicate the presence of DV patterning The regenerating spinal cord is surrounded by blastema cells, information within the mature limb that is required for proper the progenitor cells that give rise to the mesodermal tissues in growth and patterning of the regenerate (Carlson, 1974; the tail, such as dermis, cartilage and muscle. How coordinated Carlson, 1975; Holder et al., 1980). growth and patterning of the different tissue types occurs on a While the molecular mechanisms underlying spinal cord molecular level during axolotl tail regeneration is still regeneration are poorly understood, the patterning of the unknown. Spinal cord transplantation experiments have developing neural tube into distinct DV progenitor domains has established that the spinal cord harbors crucial dorsoventral been molecularly characterized in recent years (reviewed by (DV) patterning information, not only for the regenerating Bronner-Fraser and Fraser, 1997; Ericson et al., 1997a; Tanabe spinal cord but also for the surrounding tissues such as muscle and Jessell, 1996). The neural tube is subdivided into distinct and cartilage (Holtzer, 1956). Holtzer rotated a piece of tail domains, as defined by a series of homeodomain and paired spinal cord 180° about its DV axis, implanted it back into the box-containing transcription factors, with the dorsalmost mature tail and amputated the tail through the operated region. domain defined by Msx1 and 2 expression, dorsolateral cells In this situation both the spinal cord and the surrounding tissue by Pax7, and lateral domains by Pax6, while Nkx6.1 and regenerated upside down, with cartilage forming dorsally with Nkx2.2 define increasingly ventral domains. The size and respect to the whole animal, but still next to the original ventral placement of these domains is controlled by several side of the spinal cord (Holtzer, 1956). This and further work morphogens. Sonic hedgehog (Shh), a cholesterol-modified 3244 Development 132 (14) Research article

extracellular signaling factor expressed in the notochord and through the drug cyclopamine, we show that it is required not the floor plate, induces ventral neural tube cell types in a only for DV patterning of the spinal cord, but also for overall concentration-dependent manner (Briscoe et al., 1999; Ericson tail regeneration. Specifically, the proliferation of blastema et al., 1997a; Ericson et al., 1997b; Litingtung and Chiang, cells and Sox9 expression in the ventral blastema is dependent 2000; Roelink et al., 1995). The Shh gradient in the neural tube on hedgehog signaling. Therefore the induction of cartilage by is antagonized by dorsally secreted bone morphogenetic the spinal cord during tail regeneration is mediated at least in proteins (Bmps) from the epidermal ectoderm and the dorsal part through hedgehog. roof plate cells of the neural tube (Liem et al., 1995), which specify a subset of interneurons in the dorsal neural tube (Lee et al., 2000; Liem et al., 1997). Whereas Bmp4 and Bmp7 Materials and methods activate the expression of Msx1, Pax7 and Pax6 in the dorsal Axolotl care and lateral neural tube, Shh has a concentration-dependent Ambystoma mexicanum (axolotls) were bred in our facility, where they inhibitory effect on the expression of these markers (Goulding were kept at 18°C in Dresden tap water and fed daily with artemia. et al., 1993; Liem et al., 1995; Timmer et al., 2002). Low For all surgery, animals were anesthetized in 0.01% ethyl-p- concentrations of Shh block Msx1 and Pax7 expression but can aminobenzoate (Sigma). The experiments described here were elevate Pax6 expression in lateral neural tube cells. High performed on 3-cm-long larval axolotls. concentrations of Shh, however, inhibit Pax6 expression in In situ hybridization on axolotl tail cryosections and floor plate cells of the neural tube (Ericson et al., 1997b). Thus, sequences of probes used during embryogenesis, the notochord ventrally and the Axolotl tail tissue was fixed in 4% fresh paraformaldehyde (PFA) ectoderm dorsally impose DV patterning on the neural tube overnight at 4°C, washed in PBS, equilibrated in 30% sucrose and through extracellular signaling. embedded in tissue-tek (O.C.T. compound, Sakura). Cryosections 16 During development, the action of Shh and Bmps is not µm thick were mounted on Superfrost adhesive slides and dried at restricted to patterning the neural tube. These morphogens also room temperature (RT) for several hours. The sections were quickly play important roles in controlling cell proliferation, patterning washed in PBS and treated with hybridization denaturation mix (2% and cell-type specification of somite-derived cells such as the SDS, 100 mmol/l DTT in 1 PBS) for 20 minutes at RT. After three sclerotome, resulting in a coordinated patterning of the neural washes in PBS/0.1% Tween, the sections were digested with Proteinase K (2-10 µg/ml) for 5 minutes and post-fixed directly afterward with tube and its surrounding mesodermal structures. Shh mutant PFA for 10 minutes at RT. Slides were washed in PBS/Tween and mice lack vertebral columns and ribs, demonstrating that Shh incubated at RT for 15 minutes in triethanolamine with 0.25% acetic signaling from the notochord and ventral neural tube is crucial anhydride. After several washes in PBS/Tween, slides were for sclerotome development (Chiang et al., 1996). More prehybridized in hybridization buffer (50% formamide, 5 SSC, 5 specifically, Shh induces the expression of sclerotomal markers Denhardts, 750 µg/ml yeast RNA) for 1 hour at 68°C, and then such as Pax1 and Sox9 (Fan and Tessier-Lavigne, 1994; hybridized overnight at 68°C with 500 ng/ml DIG-labeled probe in Development Marcelle et al., 1999; Murtaugh et al., 1999; Zeng et al., 2002), hybridization solution. Slides were washed twice an hour at 68°C in which are essential for sclerotome development and cartilage post-hybridization solution (50% formamide, 2 SSC, 0.1% Tween) formation (Bi et al., 1999; Peters et al., 1999). Similarly, Shh and then 3 10 minutes at RT in maleic acid buffer (100 mmol/l maleic regulates myogenic precursors by positively regulating Myf5 acid pH 7.5, 150 mmol/l NaCl, 0.1% Tween). Sections were blocked in maleic acid buffer plus 10% goat serum for 1 hour at RT and then expression (Gustafsson et al., 2002). In addition to sclerotomal incubated overnight at 4°C in blocking buffer plus alkaline and myogenic markers, Shh induces proliferation of the phosphatase conjugated anti-DIG antibody (diluted 1:2000). Slides somitic mesoderm (Fan et al., 1995; Marcelle et al., 1999). Shh were washed 2 5 minutes in maleic acid buffer and 2 20 minutes also negatively regulates its own signaling by upregulation of in alkaline phosphatase buffer (100 mmol/l Tris pH 9.5, 50 mmol/l its own binding receptor Patched1 (Goodrich et al., 1996). MgCl2, 100 mmol/l NaCl, 0.1% Tween). Each slide was overlaid with Taken as a whole, this information indicates that Shh signaling filtered NBT-BCIP (Sigma) for 1-2 days at RT. The staining reaction plays diverse roles in the somite, namely proliferation, was stopped with PBS/Tween and the slides mounted in 90% glycerol. patterning and negative feedback. The interplay of all three Sense and antisense probes for in situ hybridizations were prepared may help define the shape and size of the developing from the axolotl Shh sequence (CO786463), Msx1 sequence sclerotome-derived skeletal components. (AY525844), Pax6 sequence (CO784109), Ptc1 sequence (AY887138) and Sox9 sequence (AY894689). Shh and Pax6 We wanted to investigate the molecular identity of the DV sequences were derived from EST sequences (Habermann et al., patterning information in the axolotl spinal cord, and how the 2004), while the Msx1, Ptc1 and Sox9 sequences were obtained by spinal cord communicates it to the regenerating spinal cord, RT-PCR from total embryonic RNA using degenerate primers (primer and subsequently to the surrounding blastema tissue. In order sequences and PCR conditions available upon request). to identify the molecular basis of the DV patterning information in the axolotl spinal cord, we asked whether these Pax7 antibody staining on axolotl tail cryosections well-described markers were present in the mature and/or Axolotl tails were fixed in 4% fresh paraformaldehyde (PFA) regenerating spinal cord. Here we demonstrate that Shh, Pax6, overnight at 4°C, washed in PBS, equilibrated in 30% sucrose and frozen in tissue-tek (O.C.T. compound, Sakura). Cross-sections of the Pax7 and Msx1 are expressed in their respective domains in the µ mature axolotl spinal cord as well as in the ependymal tube. tail 16 m thick were processed for immunohistochemistry with the anti-Pax7 mAB (Pax7, Developmental Studies Hybridoma Bank, This represents the first time that the molecular basis of DV Iowa, USA). A Cy5-labeled secondary antibody (Dianova, Hamburg, patterning information in the mature axolotl tissue has been Germany, http://www.dianova.com) was used at 1:200 dilution. defined. Patched1 expression further indicates that hedgehog Nuclear stainings were done with 1 µg/ml of Hoechst. To calculate signaling occurs both within the spinal cord, and in the percentage of Pax7-positive cells in the blastema, between 704 surrounding blastema cells. By blocking hedgehog signaling and 1128 blastema cells were counted in total per regenerate. Hedgehog signaling in axolotl tail regeneration 3245

Cyclopamine and agonist treatment Results Cyclopamine was purchased from Toronto Research Chemicals. Two Shh, Pax6, Pax7 and Msx1 are expressed in the hedgehog agonists in the same chemical class as described (Frank- mature and regenerating axolotl spinal cord Kamenetsky et al., 2002), but with different EC50 values, were obtained from the Curis Corp (http://www.curis.com/), and tested. In order to determine the molecular nature of DV patterning Both gave identical results with respect to their EC50 concentrations. in the axolotl spinal cord, we examined the expression of Hh-Ag1.9 is available from the Curis Corp. Unless indicated marker genes that characterize the DV axis of the developing otherwise, larval axolotls were exposed to cyclopamine and the neural tube. We chose the secreted signaling molecule Shh and hedgehog agonist directly after tail or limb amputation. the transcription factors Pax6, Pax7 and Msx1 as well- Cyclopamine-treated axolotls were kept in 20 ml water plus 600 described and distinct markers of dorsoventral neural nmol/l cyclopamine (diluted from 5 mmol/l stock solution in progenitor cell populations in the embryonic neural tube ethanol). Agonist-treated axolotls were kept in 20 ml water plus 4, µ (Echelard et al., 1993; Jostes et al., 1990; Robert et al., 1989; 40, 100, 300 nmol/l agonist (diluted from 400 mol/l stock solution Walther and Gruss, 1991). All these markers were expressed in DMSO). Control animals were kept in 20 ml water, or 20 ml water plus 0.0125% ethanol, or 20 ml water plus the agonist-equivalent in both the mature axolotl spinal cord and in the ependymal amount of DMSO, or 20 ml water plus 600 nmol/l tomatidine tube in DV domains very similar to those found in (Toronto Research Chemicals). development. Shh was expressed in the ventralmost cells of the axolotl spinal cord (the floor plate) (Fig. 1A-C); Pax6 was Cumulative BrdU labeling and anti-BrdU antibody staining expressed in the lateral spinal cord cells (Fig. 1D,E); and Pax7 Axolotl tails were amputated and treated with 600 nmol/l cyclopamine was expressed in the dorsolateral cells of the spinal cord (Fig. or equivalent amounts of ethanol. Animals were injected intra- 1F,G). In addition to the expression in the spinal cord, Pax7 peritoneally with 10 mg of BrdU (in a volume of 10 ml) every 8 hours was also expressed in the lateral edges of the blastema (Fig. starting 3 days post-amputation (dpa). Tails were fixed in 4% freshly 1G). It is likely that these Pax7-positive blastema cells made PFA 48 and 72 hours after the initial BrdU injection. represent muscle progenitor cells, as Pax7 is a known satellite Cryosections 16 µm thick were prepared and processed for antibody staining with mouse monoclonal anti-BrdU antibody directly coupled cell marker, and the Pax7-positive cells in the mature tail laid to rhodamine (Tanaka et al., 1997). Nuclear staining was performed adjacent to muscle fibers (data not shown). Msx1 was using 1 µg/ml Hoechst. The percentages of BrdU-positive ependymal expressed in the dorsalmost spinal cord, the roof plate cells cell nuclei, and BrdU-positive blastema cell nuclei ventral to the (Fig. 1H-J). In this analysis it was crucial to perform the gene ependymal tubes were calculated. The graphs in Fig. 6 represent the and protein expression analysis on tissue sections rather than mean percentage of BrdU-positive cells of 2-4 regenerates. Between whole-mount preparations. With whole mounts, spinal cord 76 and 255 cells in total were counted in ependymal tube and blastema staining was observed in the regenerate but not the mature per regenerate. tissue, presumably due to insufficient penetration of in situ probe and antibody in the mature part of the tissue, and thus Spinal cord removal from the axolotl tail

Development would have given the false impression of a regeneration- Axolotls were anesthetized and the tail was sliced open from the specific upregulation of the genes. dorsal side until the level of the spinal cord. The spinal cord was removed over the length of several segments and the tail was allowed Our gene expression analysis indicates that the putative to heal for several days. Mock operated axolotl tails were opened until progenitor cells in the mature axolotl spinal cord show an the level of the spinal cord and allowed to heal without removal of embryonic pattern of DV neural tube markers. In addition, the the spinal cord. Amputation was performed a few days after the same DV pattern is present in the ependymal tube throughout operation. axolotl tail regeneration.

Fig. 1. Shh, Pax6, Pax7 and Msx1 are expressed in the differentiated and regenerating axolotl spinal cord. All panels are cross-sections with the dorsal side up. (A-C) Shh is expressed in the floor plate of the differentiated spinal cord (A) and in the ventralmost ependymal cells 6 dpa (B,C). The overview in C shows that Shh is expressed exclusively in the spinal cord. (D,E) Pax6 is expressed in the lateral cells of the differentiated axolotl spinal cord (D) and in the lateral ependymal cells 8 dpa (E). (F,G) Pax7 is expressed in the dorsolateral cells of the differentiated spinal cord (F) and in the dorsolateral domain of the ependymal tube 6 dpa. In addition, Pax7 is expressed in lateral tail cells (F,G). (H-J) Msx1 is expressed in the roof plate of the differentiated spinal cord (H) and in the dorsalmost ependymal cells 4 and 5 dpa (I,J for overview). Panels A-E,H-J show in situ hybridizations, panels F,G antibody staining. Scale bars: 100 µm in A,B,E-G; 50 µm in C,D,H-J. 3246 Development 132 (14) Research article Hedgehog signaling is required for overall tail Hedgehog signaling is necessary for the correct regeneration establishment of DV progenitor domains in the Shh is a potent morphogen patterning the ventral half of the ependymal tube during tail regeneration spinal cord, which leads to the correct spatial organization of When we examined the ependymal tube in cyclopamine- interneurons and motoneurons during development (reviewed treated regenerates for DV patterning defects we observed by Litingtung and Chiang, 2000; Marti and Bovolenta, 2002). expansion of the dorsal spinal cord markers Pax7 and Msx1 Because Shh was expressed in the mature and regenerating into more ventral regions (compare Fig. 3A,D with Fig. 1G,I). axolotl spinal cord, we wanted to assess its function in the In cyclopamine and agonist-treated regenerates, both the Msx1 establishment of the DV identity of the regenerating tail. An and Pax7 expression domains were restored, demonstrating the interesting question for us was whether interfering with the DV rescue of the cyclopamine effect (compare Fig. 3B,E with Fig. pattern in the regenerating spinal cord would have an effect on 1G,I). Treatment of regenerating tails with the agonist alone the overall DV organization of the regenerating tail: for did not have any overall morphological effects, although the example, on the position of cartilage formation. Furthermore, regenerating tails might have been slightly bigger. We did, we wanted to examine whether Shh is necessary for ependymal however, observe an effect of the agonist alone on DV cell proliferation, as it has been shown that Shh can act as a patterning markers in the spinal cord. Even low concentrations mitogen on neural progenitor cells, both in vitro and in vivo (4 nmol/l) of agonist abolished Pax7 expression from the dorsal (Bambakidis et al., 2003; Lai et al., 2003; Machold et al., 2003; spinal cord (Fig. 3C). By contrast, Pax7-positive cells in the Palma et al., 2005). surrounding lateral blastema tissue, which presumably In order to inhibit the Shh signaling pathway during tail represent muscle progenitors, persisted in the presence of regeneration, we turned to the widely used chemical inhibitor agonist (Fig. 3C). cyclopamine, which blocks hedgehog signaling by We conclude from these results that hedgehog signaling is antagonizing the hedgehog receptor Smoothened (Chen et al., required for the correct establishment of DV progenitor 2002; Taipale et al., 2000). The drug can be easily administered domains in the regenerating axolotl spinal cord. through the axolotl water. Interestingly, we found that in the presence of cyclopamine overall axolotl tail regeneration was Patched1 is expressed in the ependymal tube as strongly inhibited (Fig. 2A-G). Wound healing and fin well as in the blastema formation occurred normally, and the ependymal tube grew to As the strongest effect of inhibiting hedgehog signaling during a limited extent, but a proper blastema did not grow (compare tail regeneration was a reduced tail blastema, we wanted to Fig. 2A-C with D-F). The rate of ependymal tube growth was know whether blastema cells directly receive the hedgehog substantially lower than control regenerates (Fig. 2G). In terms signal. We therefore examined the expression of the hedgehog of the blastema phenotype, few blastema cells had accumulated binding receptor Patched1 (Ptc1) in tail regenerates by in situ in cyclopamine-treated regenerates 4 days post-amputation hybridization. In normal regenerates Ptc1 was expressed in Development (dpa) in comparison with the control (compare Fig. 2A with ventral and lateral spinal cord cells, and in the blastema cells D). The effect became more evident at later stages of surrounding the ventral spinal cord (Fig. 4B,C; note the regeneration, when even up to 14 dpa neither cartilage nor absence of staining in the epidermis). Ptc1 itself is a target gene muscle differentiation took place in cyclopamine-treated of the hedgehog signaling pathway that is upregulated where regenerates (compare Fig. 2B,C with E,F). Cartilage and Shh signaling occurs (Goodrich et al., 1996). Agonist-treated muscle started to differentiate at 6 and 10 dpa, respectively, in regenerating tails showed increased Ptc1 expression: most or control regenerates (not shown). After 8 days of cyclopamine all of the ependymal cells and also most of the blastema cells treatment, the initial outgrowth of the ependymal tube stopped, expressed Ptc1 (Fig. 4E,F). Together these data indicate that and the tube slowly regressed over the following days (Fig. blastema cells receive the hedgehog signal directly. Although 2G). The inhibitory effect of cyclopamine on tail regeneration it is not known if other hedgehog family members are also could be observed at concentrations ranging from 600 nmol/l expressed during regeneration, the expression of Ptc1 in the to 6 µmol/l, while the same concentrations of tomatidine, a ependymal tube and the surrounding blastema tissue is closely related compound to cyclopamine that does not consistent with the regenerating spinal cord as the primary interfere with Shh signaling, did not have this effect (Fig. 2G). source of hedgehog signal. As the various concentrations of cyclopamine tested all yielded very similar results, only the lowest concentration (600 nmol/l) Hedgehog signaling is required for Sox9 expression was used for the experiments reported here. in the tail blastema Experimental evidence that cyclopamine exerts a specific Knowing that Shh signaling can occur from the ependymal inhibition of the hedgehog signaling pathway during tail tube to the surrounding blastema cells, we wanted to regeneration was the ability to rescue the phenotype with a investigate whether Shh is required for patterning the blastema hedgehog-pathway agonist. When we added a hedgehog agonist tissue. During development, Shh induces the expression of the in the same chemical class described in Frank-Kamenetsky et early cartilage marker Sox9 in the sclerotome (Tavella et al., al. (Frank-Kamenetsky et al., 2002) (see Materials and 2004; Zeng et al., 2002). We examined whether cartilage methods) together with cyclopamine, a tail with normal progenitors in the early blastema express Sox9, and whether cartilage and muscle patterning regenerated (Fig. 2H-K). this expression is controlled by hedgehog signaling. We found The inhibition of tail regeneration by cyclopamine and the that Sox9 was expressed in a defined area of the blastema rescue of this phenotype with a hedgehog-pathway agonist ventral to the spinal cord from 4 dpa onward (data not shown), strongly suggest that hedgehog signaling is required for overall which is 2 days before obvious cartilage differentiation. By tail regeneration. contrast, Sox9 expression was not detectable in cyclopamine- Hedgehog signaling in axolotl tail regeneration 3247 Development

Fig. 2. Cyclopamine treatment blocks axolotl tail regeneration and a hedgehog agonist rescues the phenotype. (A-C) Control regenerates 4, 8 and 14 dpa. The blastema cells are surrounding the ependymal tube at 4 dpa (A). Cartilage has differentiated 8 dpa (B) and muscle 14 dpa (C). (D-F) Cyclopamine-treated tail regenerates 4, 8 and 14 dpa. The fin and the ependymal tube have grown, but a blastema is not visible (compare A and D). No cartilage has differentiated 8 dpa (E) and no muscle has formed 14 dpa (F). (G) Quantification of the lengths of control and cyclopamine-treated tail regenerates over time. The length of the regenerating spinal cord is taken as a measurement for overall tail regeneration. Data points represent the average length of three regenerates; error bars are standard deviations. (H-J) Cyclopamine-treated regenerate (H), cyclopamine and agonist-treated regenerate (I), and normal tail regenerate (J) 10 dpa. The tails in I and J are indistinguishable. (K) Quantification of regenerate lengths (as in G) in indicated conditions shows that 40 nmol/l of the agonist are sufficient to rescue the cyclopamine effect, whereas 4 nmol/l are not. The dashed line in A-F and H-J marks the amputation plane. Scale bars: 0.5 mm. 3248 Development 132 (14) Research article

treated regenerates 6 dpa (Fig. 5A,B), while agonist-treated regenerates showed an increased expression domain of Sox9, and occasional dorsal blastema cells expressing the gene (Fig. 5C, arrows point to Sox9-positive cells). Despite this expanded expression of Sox9 in the agonist-treated sample, no overt cartilage differentiation was observed in the dorsal blastema, and the ventral cartilage rod appeared normal. We further examined whether hedgehog signaling is required for the putative muscle progenitors during tail regeneration. It was evident that cyclopamine-treated tail blastemas contained fewer Pax7-positive cells (compare Fig. 3A with Fig. 1G and Fig. 3B). We quantified the percentage of cells in the blastema that were Pax7-positive and found that it was reduced to approximately half the amount in cyclopamine- treated versus control regenerates (10 versus 23%; Fig. 5D). Whereas Sox9 expression seemed to be completely abolished from the cyclopamine-treated blastema, Pax7 was still expressed, but the number of Pax7-positive blastema cells was significantly reduced. Hedgehog signaling controls blastema cell proliferation rather than ependymal cell proliferation The overall morphology of cyclopamine-treated regenerates Fig. 3. Dorsal spinal cord progenitor domains are increased in indicated that the fin was normal, but the size of the blastema cyclopamine-treated and normal in rescued regenerates. (A-C) Pax7 was severely reduced (compare Fig. 2A with 2D). On cross- antibody stainings on cross-sections of cyclopamine-treated sections we observed that cyclopamine-treated regenerates had regenerate (A), rescued regenerate (B), and agonist- treated a smaller width compared with controls (compare Fig. 3A with regenerate (C). The Pax7 expression domain in the Fig. 1G and Fig. 3B, and Fig. 5A with 5B). dorsal ependymal tube is expanded ventrally in A, normal in B We examined whether the reduction of the blastema was due (compare to Fig. 1G), and absent in C (dashed line in C marks to apoptosis or a block in cell division. TUNEL staining of the ependymal tube). The arrows in A,B point to the ventral border cyclopamine and control samples were indistinguishable, of Pax7 expression in the ependymal tube. Note that the Pax7 suggesting that massive apoptosis did not account for the Development expression persists in the lateral blastema cells in C. Pax7 staining is blastema defect (data not shown). To examine cell in red and nuclear Hoechst staining in blue. (D,E) In situ hybridization of Msx1 on cross-sections of cyclopamine-treated (D) proliferation, we performed cumulative BrdU labeling for 48 and rescued regenerates (E). Msx1 expression in the ependymal tube and 72 hours, starting 3 dpa. The percentage of BrdU-positive of cyclopamine-treated regenerates is strongly expanded laterally ependymal cells and BrdU-positive ventral blastema cells was (D, compare with Fig. 1I). The rescued tails show normal Msx1 calculated in control and cyclopamine-treated regenerates at 48 expression (F). Scale bars: 100 µm. hours after the initial injection. We observed a different effect of cyclopamine on proliferation of ependymal cells versus

Fig. 4. Ependymal cells and tail blastema cells express the hedgehog receptor Ptc1. (A-F) In situ hybridization of Ptc1 on cross-sections. Sense probe shows no staining of the entire cross-section both in control (A) and agonist-treated (D) regenerates. Ptc1 is expressed in the ventral ependymal cells (dashed line marks the ependymal tube) and in the blastema, but not in the epidermis (B,C). Note that the staining in the blastema is strongest in the cells surrounding the ventral spinal cord. C is taken at twice the magnification of B. A-C is 4 dpa. Ptc1 expression is increased in agonist-treated regenerates (E,F). Now all the ependymal cells express Ptc1 and the vast majority of ventral blastema cells do (compare ventral to dorsal blastema cells). F is taken at twice the magnification of E. D-F is 5 dpa. Scale bars: 100 µm. Hedgehog signaling in axolotl tail regeneration 3249

Fig. 5. Sox9 and Pax7 expression is reduced in cyclopamine-treated blastemas. (A-C) Sox9 in situ hybridization on cross-sections of control (A), cyclopamine-treated (B) and agonist-treated (C) regenerates 6 dpa. Note the absence of staining in B. The arrows in C point to Sox9- positive dorsal blastema cells. (D) Quantification of Pax7-positive blastema cells in control and cyclopamine-treated regenerates 6 dpa. Columns represent the mean percentage of Pax7-positive blastema cells of three regenerates (total number of counted cells per regenerate is between 704 and 1128). Error bars indicate standard deviations. The t-test value is 0.0007. Scale bar: 100 µm in A-C.

ventral blastema cells (compare Fig. 6A with 6B). Whereas expected defects in anteroposterior (AP) digit patterning as in cyclopamine treatment had only a minor effect on the fraction the developing limbs of the Shh knockout mouse (Chiang et of proliferating ependymal cells (from 99 to 86%; Fig. 6A), it al., 1996), and as previously observed for axolotl limb had a strong, statistically significant inhibitory effect on the regeneration (Roy and Gardiner, 2002). We conclude that Shh fraction of proliferating ventral blastema cells, from 95 to 56% has different effects on limb versus tail regeneration. Whereas (Fig. 6B). This inhibitory effect was stable over time, as we cyclopamine-treated tails showed a profound effect on both observed the same decrease of BrdU incorporation at 72 hours, growth and patterning of the regenerate, cyclopamine-treated indicating that all proliferating cells had incorporated BrdU. limbs were affected only in the AP patterning of the digits. We conclude that hedgehog signaling controls the proliferation These data, together with our previous data, further indicate of approximately 40% of ventral tail blastema cells. This that the block in tail regeneration in response to cyclopamine number could be an underestimate, because we could have represents a specific inhibition of the hedgehog signaling inadvertently included some fin cells (that regenerate normally) pathway.

Development in the analysis. Ectopic activation of hedgehog signaling in the Shh has distinct activities on the limb versus the tail absence of the spinal cord is not sufficient for tail blastema regeneration To address whether the cyclopamine effect on tail blastema cell It was previously shown that tail regeneration is absolutely proliferation might represent a nonspecific effect on cell dependent on the presence of the spinal cord at the plane of division, we examined whether cyclopamine had a distinct amputation. Removal of the spinal cord from the distal tip of effect on the limb blastema. When we treated regenerating limb the tail and subsequent tail amputation blocks growth until the blastemas with cyclopamine we obtained limb regenerates of spinal cord regenerates back (Donaldson and Wilson, 1975; normal length but lacking digits (Fig. 7), consistent with the Holtzer et al., 1955). Due to the striking similarity between

A Ependymal cell proliferation B Blastema cell proliferation 100 100

80 80

60 60 ethanol ethanol 40 40 cyclopamine cyclopamine

20 20 % of BrdU positive cells % of BrdU 0 positive cells % of BrdU 0 48 72 48 72 hours after first BrdU injection hours after first BrdU injection

Fig. 6. Hedgehog signaling controls blastema cell proliferation. (A,B) Cumulative BrdU labeling of ependymal cells (A) and ventral blastema cells (B). Each column represents the mean percentage of BrdU-positive cells of 2-4 regenerates at indicated time intervals of BrdU labeling. Between 76 and 255 ependymal and blastema cells were counted in total per regenerate. Error bars are standard deviations. The t-test values are 0.005 at 48 hours and 0.212 at 72 hours in A, and 0.003 at 48 hours and 0.002 at 72 hours in B. 3250 Development 132 (14) Research article

is important for the correct propagation of the domains during regeneration. (1) DV rotation of the mature spinal cord resulted in a rotated orientation of the regenerated spinal cord, indicating that the DV patterning information in the regenerate comes from the mature spinal cord. (2) Lineage tracing experiments indicated that the DV progenitor cell domains in the axolotl spinal cord are not transmitted to the regenerating tail by simple lineage restriction, as 30% of clones from a single ependymal cell spanned multiple DV domains (L. Fig. 7. Cyclopamine treatment of the regenerating axolotl limb does Mchedlishvili, A. Telzerow, H. H. Epperlein and E.M.T., not affect blastema growth but leads to digit loss. (A) The unpublished). (3) The evidence presented in this paper regenerated control limb 15 dpa. (B) The regenerated cyclopamine- indicates that Shh is expressed in the ventral floor plate of the treated limb structure 15 dpa. The arrow in B points to the mature and regenerating spinal cord. Inhibition of hedgehog regenerated rod of cartilage. The dashed line in A and B marks the signaling during tail regeneration caused an expansion of amputation plane. Scale bar: 0.5 mm. dorsal neural progenitor domains, while activation of hedgehog signaling induced a reduction of dorsal progenitor domains. This indicates that ventral Shh expression in the regenerating spinal cord removal and the cyclopamine effect on tail spinal cord controls DV progenitor cell identity during tail regeneration, we wanted to test whether hedgehog signaling is regeneration. How the Shh expression domain itself is the sole spinal cord signal required for blastema growth and maintained in the mature spinal cord and established in the thus tail regeneration. We removed the spinal cord from the tip regenerating tube is not yet clear. Taken together, these data of the axolotl tail, amputated through the tail devoid of spinal strongly indicate that, although during development the initial cord, and treated animals with the hedgehog agonist. No DV patterning of the neural tube is imposed from structures regeneration occurred in tails without spinal cord, either in the outside the neural tube, in regeneration the source of ventral presence or absence of agonist (Fig. 8A-C). This result Shh signaling comes from within the regenerating spinal cord. indicates that hedgehog signaling is not sufficient to rescue tail regeneration in the absence of the spinal cord. At least one The molecular circuitry controlling DV spinal cord other factor must exist in the spinal cord that is required for patterning during regeneration tail regeneration. In these experiments, we tested the role of hedgehog signaling by bathing the animals in a uniform concentration of the inhibitor, cyclopamine or the agonist, Hh-Ag1.9. Cyclopamine Discussion alone caused ventral expansion of dorsal neural progenitor Development We have examined the DV patterning information that is markers such as Pax7, while agonist alone caused the present in the mature axolotl spinal cord and its transmission disappearance of the Pax7 domain, presumably representing a to the regenerate. This work has uncovered a role for hedgehog severe ventralization of the ependymal tube. Interestingly, the signaling, not only in DV patterning of the spinal cord and combination of cyclopamine and agonist restored a relatively surrounding tissues such as cartilage, but also in blastema cell normal Pax7 domain, and indeed, normal growth and DV proliferation. We have shown that Shh, Pax6, Pax7 and Msx1 patterning of the entire tail. On the surface it may seem were expressed in the differentiated axolotl spinal cord in DV surprising that restoration of normal patterning occurs in domains similar to those found in development, and that this response to uniform application of an inhibitor and agonist to expression persisted in the ependymal tube throughout tail a morphogen that clearly acts in a concentration-dependent regeneration. This represents the first time that the patterning manner (Briscoe et al., 1999; Ericson et al., 1997a; Ericson et information present in the urodele mature tissue and required al., 1997b; Roelink et al., 1995). It should be understood, for regeneration has been localized on a molecular and cellular however, that these pharmacological treatments are level. One question that arises is how these domains are superimposed on the normal expression of endogenous ‘propagated’ along the growing ependymal tube. Several morphogens such as Shh and presumably Bmp family pieces of evidence suggest that signaling within the spinal cord members. For example, we did not see an alteration of Shh

Fig. 8. Hedgehog signaling is not sufficient for tail regeneration in the absence of the spinal cord. (A) Mock operated axolotl tail shows normal 7-day regenerate. (B) Control tail with spinal cord removed does not regenerate. (C) Agonist-treated tail without spinal cord also does not regenerate. Arrows point to the distal tip of the spinal cord in all panels. The dashed line marks the amputation plane. Scale bar: 0.5 mm. Hedgehog signaling in axolotl tail regeneration 3251

expression in the presence of cyclopamine. This means that (Fan and Tessier-Lavigne, 1994; Marcelle et al., 1999; endogenous gradients of hedgehog and Bmp are probably still Murtaugh et al., 1999; Tavella et al., 2004; Zeng et al., 2002). functioning in the face of uniform chemical agents. It is In the blastema, the location of Sox9 expression with respect therefore likely that the inhibitor/agonist co-treatment to the regenerating spinal cord is distinct from that during uniformly balances out inhibition and activation of the development, as it appears ventral to the ependymal tube rather hedgehog pathway, allowing the endogenous concentration- than in lateral sclerotomal cells. Although regeneration does dependent signaling to be manifested. not proceed through a morphologically distinct somite, it is In terms of establishing the various DV progenitor cell very likely, however, that the molecular signaling pathway domains within the spinal cord, a relatively detailed leading to cartilage formation in the two contexts are the same. understanding has been gained in embryonic studies, and we Second, the hedgehog agonist could induce ectopic Sox9 assume that the same signaling networks are implemented expression in dorsal regions of the blastema. The fact that only during regeneration. In particular, Briscoe et al. (Briscoe et al., isolated dorsal blastema cells expressed Sox9, rather than 2000) have suggested a model to explain how Shh signaling massive formation of cartilage throughout the blastema in from the floor plate could result in the establishment of distinct agonist-treated regenerates, is probably due to the inhibitory neural progenitor domains along the DV axis of the neural tube. role of molecules such as Bmps in the dorsal regenerate that Graded Shh signaling results in the definition of two distinct would antagonize the agonist effect. types of molecular domains. The expression of so-called class I homeodomain proteins such as Pax7, Irx3, Dbx1, Dbx2 and The role of hedgehog signaling in blastema cell Pax6 (found in dorsolateral regions) are repressed by Shh proliferation signaling, while expression of class II homeodomain proteins A striking aspect of our results is the profound dependence of including Nkx6.1 and Nkx2.2 are activated by Shh signals. tail regeneration on hedgehog signaling. BrdU labeling Cross-repressive interactions between class I and class II indicated at least a 40% reduction in cycling blastema cells. homeodomain proteins, such as those between Pax6 and This result probably represents an underestimate, because it is Nkx2.2 (Briscoe et al., 2000), establishes, refines and stabilizes difficult to distinguish the cycling fin cells from the blastema the progenitor cell domains. Although a specific class II protein cells due to lack of a blastema cell marker. We favor the idea that represses Pax7 has not been identified yet, presumably that sonic hedgehog is a direct mitogen for blastema cells, as additional class II proteins may exist (Briscoe et al., 2000). the patched receptor is expressed in the blastema, although it Therefore, in our case the ventral expansion of Pax7 in is possible that hedgehog expression in the regenerate may be cyclopamine-treated regenerates is probably due both to an necessary for the expression of a blastema cell mitogen. For increase in Pax7 expression stemming from reduced hedgehog example, in the limb sonic hedgehog expression upregulates signaling, and a decrease in the level of class II proteins that Fgf4 in the apical ectodermal ridge to promote limb bud require hedgehog signaling for their expression and that act by outgrowth (Laufer et al., 1994; Niswander et al., 1994). We Development restricting Pax7 expression to a dorsal domain. Conversely, have tested the role of signaling through the Fgfr1 in tail reduction of Pax7 in ependymal tubes of hedgehog-agonist- regeneration and found that it cannot account for the effect of treated regenerates might be due to both an increase in hedgehog. While chemical inhibition of Fgfr1 signaling during hedgehog signals and in the level of class II proteins that tail regeneration via the pharmacological inhibitor SU5402 subsequently repress Pax7 in the dorsal tube. initially slowed down regeneration slightly, the regenerated During development, Bmps in the dorsal ectoderm and roof tails showed no other phenotype and grew to a normal length plate are crucial morphogens for DV neural tube patterning. (data not shown) – a phenotype quite distinct from hedgehog We surmise that Bmp4 and Wnt3a are expressed in the dorsal inhibition. It appears, however, that hedgehog is not the sole axolotl spinal cord. Although we could detect Bmp4 and factor required for blastema cell proliferation, as the hedgehog Wnt3a in tail blastema RNA by RT-PCR, attempts to localize agonist could not rescue the blastema growth defect produced Bmp4 and Wnt3a by in situ hybridization or phospho-Smad1 by spinal cord removal. immunohistochemistry have so far been unsuccessful. The presence of Msx1, a known downstream target of Bmp4 (Liem Concluding remarks et al., 1995; Timmer et al., 2002), in the axolotl dorsal spinal Our study also has implications for understanding the origin cord suggests the presence of Bmp signaling within the spinal and fate of blastema cells. As Shh is already expressed in the cord. mature spinal cord, the tail blastema cells receive signals that direct them to specific cell fates as soon as they are born. A The role of hedgehog signaling in patterning the tail naive blastema cell may therefore be extremely difficult to blastema detect. Although the ventral blastema cells behave similarly to In addition to the role of hedgehog signaling in patterning the sclerotome, it is not clear if an early blastema cell that responds regenerating spinal cord, we have demonstrated that hedgehog to the Shh signal is equivalent to an early somite cell, a is also required for patterning the surrounding blastema tissue. presomitic mesoderm cell, or is a completely distinctive cell The early cartilage marker Sox9 was not expressed in type. It is possible, for example, that the blastema cell has more cyclopamine-treated animals. We favor the idea that this fates available to it than a typical sclerotomal cell. reflects a requirement of hedgehog to induce Sox9 expression Furthermore, it is unclear if the Sox9-expressing blastema cells rather than complete absence of Sox9-expressing cells in the derive solely from sclerotomal derivatives in the mature tissue, blastema, for several reasons. First, during development, Shh or whether different tissue types can contribute blastema cells signaling from the notochord and neural tube induces Pax1, that are induced to express Sox9. Echeverri and Tanaka Pax9 and Sox9 in the sclerotome, the precursors for cartilage (Echeverri and Tanaka, 2002) showed that cells can migrate 3252 Development 132 (14) Research article

from the spinal cord and contribute to cartilage during tail activity in amputated newt tails locally deprived of the spinal cord regeneration, indicating that cartilage precursors have diverse (including a note on effects of amputation level on mitosis). J. Exp. Zool. origins. Specific labeling of different cell types in the mature 191, 9-24. Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., tissue and long-term lineage tracing will be required to fully McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member address this issue. of a family of putative signaling molecules, is implicated in the regulation It is noteworthy that the role of hedgehog signaling during of CNS polarity. Cell 75, 1417-1430. axolotl limb regeneration is clearly different from its role in Echeverri, K. and Tanaka, E. M. (2002). Ectoderm to mesoderm lineage switching during axolotl tail regeneration. Science 298, 1993-1996. tail regeneration. In the limb blastema, Shh controlled AP digit Ericson, J., Briscoe, J., Rashbass, P., van Heyningen, V. and Jessell, T. M. formation and did not severely inhibit blastema outgrowth or (1997a). Graded sonic hedgehog signaling and the specification of cell fate cartilage formation. This indicates that blastema cells probably in the ventral neural tube. Cold Spring Harb. Symp. Quant. Biol. 62, 451- have region-specific identities that allow them to respond to 466. inductive cues in distinct ways. Presumably this region-specific Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M. and Briscoe, J. (1997b). Pax6 controls identity is maintained in the mature tissue and inherited by progenitor cell identity and neuronal fate in response to graded Shh blastema cells, although it is possible that such identity is signaling. Cell 90, 169-180. positively reinforced during regeneration, and that in certain Fan, C. M. and Tessier-Lavigne, M. (1994). Patterning of mammalian cases, this identity could be reversed. somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell 79, 1175-1186. The maintenance of patterning information in the mature Fan, C. M., Porter, J. A., Chiang, C., Chang, D. T., Beachy, P. A. and tissue may be a central feature of regenerative ability. Adult Tessier-Lavigne, M. (1995). Long-range sclerotome induction by sonic mouse and chick spinal cord tissue does not maintain the hedgehog: direct role of the amino-terminal cleavage product and markers examined here, and this may represent a block for modulation by the cyclic AMP signaling pathway. Cell 81, 457-465. regeneration (Fu et al., 2003; Yamamoto et al., 2001). Frank-Kamenetsky, M., Zhang, X. M., Bottega, S., Guicherit, O., Wichterle, H., Dudek, H., Bumcrot, D., Wang, F. Y., Jones, S., Shulok, Interestingly, injury of the mouse spinal cord did result in the J. et al. (2002). Small-molecule modulators of Hedgehog signaling: appearance of Pax7-positive cells in the parenchyma of the identification and characterization of Smoothened agonists and antagonists. dorsal horn (Yamamoto et al., 2001). These Pax7-positive cells J. Biol. 1, 10. co-stained with nestin, indicating that they may have the Fu, H., Qi, Y., Tan, M., Cai, J., Hu, X., Liu, Z., Jensen, J. and Qiu, M. (2003). Molecular mapping of the origin of postnatal spinal cord ependymal capacity to act as progenitor cells. Such observations suggest cells: evidence that adult ependymal cells are derived from Nkx6.1+ ventral the possibility that mammals harbor a latent capacity to re- neural progenitor cells. J. Comp. Neurol. 456, 237-244. induce important aspects of cell patterning after injury. The Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. and Scott, comparison of patterning marker expression in spinal cord M. P. (1996). Conservation of the hedgehog/patched signaling pathway from progenitor cells (and other tissues) in different species may be flies to mice: induction of a mouse patched gene by Hedgehog. Genes Dev. 10, 301-312. an important dimension of understanding the regenerative Goulding, M. D., Lumsden, A. and Gruss, P. (1993). Signals from the ability. notochord and floor plate regulate the region-specific expression of two Pax Development genes in the developing spinal cord. Development 117, 1001-1016. We are grateful to Heino Andreas for dedicated axolotl care, and to Gustafsson, M. K., Pan, H., Pinney, D. F., Liu, Y., Lewandowski, A., Veronique Dubreuil for help with the in situ hybridization protocol on Epstein, D. J. and Emerson, C. P., Jr (2002). Myf5 is a direct target of cryosections, and for advice on the manuscript. long-range Shh signaling and Gli regulation for muscle specification. Genes Dev. 16, 114-126. Habermann, B., Bebin, A. G., Herklotz, S., Volkmer, M., Eckelt, K., Pehlke, K., Epperlein, H. H., Schackert, H. K., Wiebe, G. and Tanaka, References E. M. (2004). An Ambystoma mexicanum EST sequencing project: analysis Bambakidis, N. C., Wang, R. Z., Franic, L. and Miller, R. H. (2003). Sonic of 17,352 expressed sequence tags from embryonic and regenerating hedgehog-induced neural precursor proliferation after adult rodent spinal blastema cDNA libraries. Genome Biol. 5, R67. cord injury. J. Neurosurg. Spine 99, 70-75. Holder, N., Tank, P. W. and Bryant, S. V. (1980). Regeneration of Bi, W., Deng, J. M., Zhang, Z., Behringer, R. R. and de Crombrugghe, B. symmetrical forelimbs in the axolotl, Ambystoma mexicanum. Dev. Biol. (1999). Sox9 is required for cartilage formation. Nat. Genet. 22, 85-89. 74, 302-314. Briscoe, J., Sussel, L., Serup, P., Hartigan-O’Connor, D., Jessell, T. M., Holtzer, H., Holtzer, S. and Avery, G. (1955). An experimental analysis of Rubenstein, J. L. and Ericson, J. (1999). Homeobox gene Nkx2.2 and the development of the spinal column. IV. Morphogenesis of tail vertebrae specification of neuronal identity by graded Sonic hedgehog signalling. during regeneration. J. Morphol. 96, 145-168. Nature 398, 622-627. Holtzer, S. W. (1956). The inductive activity of the spinal cord in urodele tail Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). regeneration. J. Morphol. 99, 1-34. A homeodomain protein code specifies progenitor cell identity and neuronal Jostes, B., Walther, C. and Gruss, P. (1990). The murine paired box gene, fate in the ventral neural tube. Cell 101, 435-445. Pax7, is expressed specifically during the development of the nervous and Bronner-Fraser, M. and Fraser, S. E. (1997). Differentiation of the vertebrate muscular system. Mech. Dev. 33, 27-37. neural tube. Curr. Opin. Cell. Biol. 9, 885-891. Lai, K., Kaspar, B. K., Gage, F. H. and Schaffer, D. V. (2003). Sonic Carlson, B. M. (1974). Morphogenetic interactions between rotated skin cuffs hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. and underlying stump tissues in regenerating axolotl forelimbs. Dev. Biol. Nat. Neurosci. 6, 21-27. 39, 263-285. Laufer, E., Nelson, C. E., Johnson, R. L., Morgan, B. A. and Tabin, C. Carlson, B. M. (1975). The effects of rotation and positional change of stump (1994). Sonic hedgehog and Fgf-4 act through a signaling cascade and tissues upon morphogenesis of the regenerating axolotl limb. Dev. Biol. 47, feedback loop to integrate growth and patterning of the developing limb bud. 269-291. Cell 79, 993-1003. Chen, J. K., Taipale, J., Cooper, M. K. and Beachy, P. A. (2002). Inhibition Lee, K. J., Dietrich, P. and Jessell, T. M. (2000). Genetic ablation reveals of Hedgehog signaling by direct binding of cyclopamine to Smoothened. that the roof plate is essential for dorsal interneuron specification. Nature Genes Dev. 16, 2743-2748. 403, 734-740. Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, Liem, K. F., Jr, Tremml, G., Roelink, H. and Jessell, T. M. (1995). Dorsal H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in differentiation of neural plate cells induced by BMP-mediated signals from mice lacking Sonic hedgehog gene function. Nature 383, 407-413. epidermal ectoderm. Cell 82, 969-979. Donaldson, D. J. and Wilson, J. L. (1975). Dedifferentiation and mitotic Liem, K. F., Jr, Tremml, G. and Jessell, T. M. (1997). A role for the roof Hedgehog signaling in axolotl tail regeneration 3253

plate and its resident TGFbeta-related proteins in neuronal patterning in the dorsal spinal cord. Cell 91, 127-138. Litingtung, Y. and Chiang, C. (2000). Control of Shh activity and signaling in the neural tube. Dev. Dyn. 219, 143-154. Machold, R., Hayashi, S., Rutlin, M., Muzumdar, M. D., Nery, S., Corbin, J. G., Gritli-Linde, A., Dellovade, T., Porter, J. A., Rubin, L. L. et al. (2003). Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron 39, 937-950. Marcelle, C., Ahlgren, S. and Bronner-Fraser, M. (1999). In vivo regulation of somite differentiation and proliferation by Sonic Hedgehog. Dev. Biol. 214, 277-287. Marti, E. and Bovolenta, P. (2002). Sonic hedgehog in CNS development: one signal, multiple outputs. Trends Neurosci. 25, 89-96. Murtaugh, L. C., Chyung, J. H. and Lassar, A. B. (1999). Sonic hedgehog promotes somitic chondrogenesis by altering the cellular response to BMP signaling. Genes Dev. 13, 225-237. Niswander, L., Jeffrey, S., Martin, G. R. and Tickle, C. (1994). A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371, 609-612. Palma, V., Lim, D. A., Dahmane, N., Sanchez, P., Brionne, T. C., Herzberg, C. D., Gitton, Y., Carleton, A., Alvarez-Buylla, A. and Ruiz i Altaba, A. (2005). Sonic hedgehog controls stem cell behavior in the postnatal and adult brain. Development 132, 335-344. Peters, H., Wilm, B., Sakai, N., Imai, K., Maas, R. and Balling, R. (1999). Pax1 and Pax9 synergistically regulate vertebral column development. Development 126, 5399-5408. Robert, B., Sassoon, D., Jacq, B., Gehring, W. and Buckingham, M. (1989). Hox-7, a mouse homeobox gene with a novel pattern of expression during embryogenesis. EMBO J. 8, 91-100. Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P. A. and Jessell, T. M. (1995). Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81, 445-455. Roy, S. and Gardiner, D. M. (2002). Cyclopamine induces digit loss in regenerating axolotl limbs. J. Exp. Zool. 293, 186-190. Taipale, J., Chen, J. K., Cooper, M. K., Wang, B., Mann, R. K., Milenkovic, L., Scott, M. P. and Beachy, P. A. (2000). Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406, 1005-1009. Development Tanabe, Y. and Jessell, T. M. (1996). Diversity and pattern in the developing spinal cord. Science 274, 1115-1123. Tanaka, E. M., Gann, A. A., Gates, P. B. and Brockes, J. P. (1997). Newt myotubes reenter the cell cycle by phosphorylation of the retinoblastoma protein. J. Cell. Biol. 136, 155-165. Tavella, S., Biticchi, R., Schito, A., Minina, E., Di Martino, D., Pagano, A., Vortkamp, A., Horton, W. A., Cancedda, R. and Garofalo, S. (2004). Targeted expression of SHH affects chondrocyte differentiation, growth plate organization, and Sox9 expression. J. Bone Miner. Res. 19, 1678-1688. Timmer, J. R., Wang, C. and Niswander, L. (2002). BMP signaling patterns the dorsal and intermediate neural tube via regulation of homeobox and helix-loop-helix transcription factors. Development 129, 2459-2472. Walther, C. and Gruss, P. (1991). Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113, 1435-1449. Yamamoto, S., Nagao, M., Sugimori, M., Kosako, H., Nakatomi, H., Yamamoto, N., Takebayashi, H., Nabeshima, Y., Kitamura, T., Weinmaster, G. et al. (2001). Transcription factor expression and Notch- dependent regulation of neural progenitors in the adult rat spinal cord. J. Neurosci. 21, 9814-9823. Zeng, L., Kempf, H., Murtaugh, L. C., Sato, M. E. and Lassar, A. B. (2002). Shh establishes an Nkx3.2/Sox9 autoregulatory loop that is maintained by BMP signals to induce somitic chondrogenesis. Genes Dev. 16, 1990-2005.